Method and device for determining local distribution of a measuring parameter

ABSTRACT

A system and method for determining the local distribution of measurement variables being measured relative to or present in a predefined measurement area of a biological sample wherein in a first optical measuring process the local distribution of a first of the measurement variables is determined using a sensor film applied to the measurement area and including a luminescence indicator reacting to the first measurement variable by a change of luminescence decay time. The luminescence decay time or a quantity derived therefrom is recorded by an imaging technique as a function of the first measurement variable. In a second optical measuring process the local distribution of a second of the measurement variables is determined simultaneously or immediately following, using an imaging technique which is effective through the sensor film of the first optical measuring process.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a method for determining the local distributionof a quantity to be measured relative to or present in a predefinedmeasurement area of a biological sample, preferably the surface of anorgan or the epidermis, where in a first measuring process fordetermining a first measurement variable a sensor film with aluminescence indicator reacting to this variable by a change of at leastone optical characteristic is applied to the measurement area and thefirst measurement variable is detected by imaging means, as well as to asystem for implementation of this method.

2. The Prior Art

For a variety of medical applications, especially in the diagnosticsector, it is of prime importance that the local distribution of ameasurement variable over the surface of an organ, such as the humanskin, or the distribution of the flow rate of a given substance throughan interface, should be determined. Besides, such measured results areuseful in checking and controlling methods of medical therapy.

Tissue oxygenation, for example, is an important parameter in diagnosingdiseases resulting from disturbed microcirculation. Oxygen supply isdetermined on the one hand by the perfusion properties of the blood, andon the other hand by transcutaneous transport properties and oxygenconsumption in the skin. In addition, oxygen supply is partly effectedby oxygen absorption from the environment, an activity known ascutaneous respiration. Problems occur when several parametersinfluencing the medically relevant variable to different degrees, are tobe detected simultaneously.

Determining oxygen concentration or transcutaneous oxygen partialpressure (tcPO₂) by means of skin electrodes is a technique well knownin the art, as is the use of optical sensors based on fluorescencequenching. The latter do not consume oxygen during measuring, which isan advantage over the use of electrodes.

Another known technique is the so-called FLIM (fluorescence lifetimeimaging) process using optical sensors on the basis of fluorescencequenching, where a sensor membrane carrying a suitable luminescenceindicator is attached to the skin region to be examined. Oxygen diffusedthrough the skin surface enters the sensor membrane, and fluorescencequenching resulting therefrom may be analyzed by the detector system.

In this context apparatus and method for measuring tissue oxygenationare described in U.S. Pat. No. 5,593,899, where oxygen-dependentquenching of a fluorescence indicator is used for measurement. Theoxygen supply of the skin is determined by applying a luminescent probewithin a skin cream to a suitable area of the skin, and covering thatarea by an oxygen-impermeable film. The optical means for excitation ofthe indicator and detection of the respective radiation is encased in ahousing whose transparent cover is directly placed over theO₂-impermeable film. An interference filter and a photodiode are addedto this set-up. The luminescent probe is subject to excitation radiationfrom a modulated radiation source via optical fiber guides. The aboveset-up is suitable only for integral measuring over the entire areacovered by the optical equipment. It is impossible with this system,however, to obtain accurate information on boundary regions betweensufficiently oxygenated and inadequately oxygenated skin regions.

The device disclosed in EP 0 516 610 B1 can be used for measuring notonly oxygen concentration, but also oxygen flow through an interface,for example, a skin area. The sensor layer of the device to beassociated with the interface offers a known, finite resistance to theoxygen flow to be measured, and is provided with at least one opticalindicator determining oxygen concentration on one side of the sensorlayer. From the concentration value measured on one side of the sensorlayer and known beforehand on the other side (ambient air) the materialflow through the interface is determined. According to a variant of theinvention the sensor layer may be scanned a really by means of animaging system (CCD), so that local distribution of oxygen flow oroxygen concentration may be measured.

Further ideas and measured results regarding local distribution ofoxygen flow and subcutaneous oxygen concentration, as well as a proposalfor a measuring process by imaging means are disclosed in the paper“Fluorescence Lifetime Imaging of the Skin PO₂: Instrumentation andResults” in Advances in Experimental Medicine and Biology, Vol. 428, pp605–611 (1997), published by Plenum Press N.Y. The paper describes asensor membrane for measuring transcutaneous oxygen concentration, whichcomprises an optical insulating layer facing the skin surface, a sensinglayer with a luminescence indicator with O₂-sensitive decay time, and asupporting layer that is impermeable to oxygen. Further described is amembrane for measuring oxygen flow, which differs from the above sensormembrane by featuring a diffusion barrier with known oxygen permeabilityinstead of the O₂-impermeable layer. For the purpose of measurement thesensor membrane is applied to the measuring surface, for example, a skinarea. The measuring process employs a modulation technique, where theLEDs emitting excitation radiation in the direction of the sensormembrane are actuated by a square-wave generator and emit square-wavemodulated excitation radiation. The emission radiation emitted by thesensor membrane is detected by a CCD camera with modulated amplificationand passed on pixel by pixel to a computing unit for image-processing.The oxygen distribution measured in a polymeric layer is documented asan image of the variations in oxygen distribution over an area of theskin.

Other imaging processes for measurement by means of phase fluorometryare described in U.S. Pat. No. 5,485,530.

A complete estimate of the oxygen supply of a certain area of the skinor surface of an organ can only be made if the oxygen status of theinspected region is complemented by information on blood supply andperfusion rate.

A number of well-advanced methods and devices are at disposal forobtaining the necessary information on perfusion, such as Laser-Dopplerflow measurements (see U.S. Pat. No. 4,476,875), by means of which localperfusion of the blood vessels may be examined on the basis of thefrequency shift of radiation emitted by a laser lightsource. It wouldalso be possible to expand emission radiation by suitable optical means,or measurement areas could be scanned step by step with the use of alaser beam, so that a picture will be obtained of the local distributionof the perfusion rate. Such methods have become known as Laser-Dopplerimaging processes (LDI). The result of a Laser-Doppler measurement willdepend on the velocity and number of red blood cells scattering thelaser light.

In U.S. Pat. No. 4,862,894, for example, a system for analyzing thebloodstream in an area of the skin is described, where a laser beam isused which is expanded by a cylindrical lens. In one variant the skinsurface is scanned by the laser line by line, and a two-dimensionalimage is detected of how the flow velocity of the blood is distributed.Another method and apparatus for measuring fluids in motion is describedin U.S. Pat. No. 5,361,769, where the area of a specimen is scanned by aLaser-Doppler imaging process. Via a lens combination in the laser beamvarying object distances are compensated, thus increasing measuringaccuracy.

So far it has not been possible to obtain satisfactory measured resultson the oxygen supply of an organ or a skin area as the quantities to bemeasured usually vary at a rate that is faster than the rate at whichthe different measuring devices or processes required therefor can besuccessively employed in the skin area to be analyzed. A further problemis presented by the heterogeneity of the area to be measured, e.g., thesurface of the skin, so that point measurements such as electrode orLaser-Doppler flow measurements are not successful whilst imagingsystems and processes such as FLIM and LDI can only be used in adjacentsites or one after the other. For example, when tests on one and thesame skin area switch from an FLIM to LDI system, this will take toolong for parameters like perfusion and oxygen status, which frequentlychange within seconds, thus preventing meaningful measurement. Whendevices have to be exchanged, it will be difficult to reposition themprecisely, so that measured areas are likely to vary slightly.

Using previous measuring systems as a basis, it is the object of thisinvention to propose a method and apparatus for determining localdistribution of several measurement variables in a predefinedmeasurement area of a biological sample, which should enable the user toobtain information on physiological parameters and their localization.

SUMMARY OF THE INVENTION

According to the invention this object is achieved by providing that forsimultaneous or immediately following determination of the localdistribution of at least one further variable in the same measurementarea a second optical measuring process is employed which is effectivethrough the sensor film.

A system in accordance with the invention for implementation of themethod is characterized by a second optical measuring device for whoseexcitation and emission radiation the sensor film of the first opticalmeasuring device is transparent, so that two independent opticalmeasuring means will cover the same measurement area. The twomeasurements may be taken simultaneously or in such rapid successionthat the physiological parameters will remain largely unchanged in thisshort time period.

An essential feature of the invention is that the local distribution ofat least two parameters or measurement variables of a biological sampleis determined by two independent optical measuring processes or systems,in order to improve diagnostic findings. One measuring process is basedon luminescence-optical determination of a parameter using a sensormembrane with a luminescence indicator incorporated therein, while forthe second or further simultaneous, optical measuring processes thesensor membrane or sensor film must be sufficiently transparent topermit optical measurement with satisfactory signal yield.

Advantageously, a luminescence-optical method should be used as firstmeasuring process, in which luminescence decay time or a quantityderived therefrom as a function of a first measurement variable isrecorded by an imaging technique. The main advantage of determiningdecay time is that the measurement will become independent of the locallight intensity of the sensor film. This will permit full lighting evenof strongly curved surfaces of the skin or some other organ by means ofa simple lighting set-up without the need for homogeneous excitation ofthe luminescent indicator. Care should be taken, however, thatsufficient luminescence be provided in each area of the sensor membranefor decay time measurement. Since decay time measurement does not dependon object distance, and the propagation time of the light from objectsurfaces at varying distances is negligible, decay time may bedetermined with sufficient accuracy for measurement surfaces of anycurvature. Thus a major demand will be fulfilled with regard totrouble-free and safe measuring of the arms and legs or other curvedskin areas of a patient. When a luminescence signal is detected aspecial CCD camera is operated such that the information contained inevery pixel may be brought into relationship with the decay time at therespective measurement site corresponding to the pixel. Alternatively, aCMOS sensor may be used instead of the CCD camera.

As first quantity to be measured local distribution of oxygenconcentration (tcPO₂), and preferably transcutaneous oxygenconcentration, or local distribution of oxygen flow (O₂-flux) throughthe organ surface, preferably the skin surface, may be determined.

It would also be possible to determine local distribution of CO₂concentration or CO₂ flow as first measurement variable.

If suitable luminophores are used the luminescence-optical measuringprocess is well suited for determining local distributions oftemperature, glucose concentration, or an ionic concentration, such asthe pH level.

As a second measuring process a Laser Doppler imaging process (LDIprocess) may be used by means of which the perfusion rate in themeasurement area is determined. To obtain strong temporal and localcorrelation for perfusion rate and oxygen status of a biological sample,both measurements must be taken practically simultaneously within oneand the same sample area. The high local resolution of modern LDIsystems (<100 μm) can be well combined with a fluorescence lifetimeimaging (FLIM) technique, thus giving excellent diagnostic results.

The properties of the measurement process will be discussed in moredetail below, with reference to a typical application, i.e., oxygensupply of the human skin. It is to be appreciated that this will put norestriction on the overall scope of the invention.

A major advantage of the invention is that both local oxygenconcentration (first parameter) in the sensor film (and thus in thebiological sample of interest) and perfusion in the same area (secondparameter) are determined, recorded by imaging and assessed bycomparison, with strong temporal and local correlation in an extendedmeasurement area. As the local oxygen concentration in the indicatorlayer of the sensor film is known, O₂ partial pressure at the skinsurface or O₂-flux may be computed in dependence of O₂ diffusionproperties of the supporting membrane and allowing for barometricpressure. From the local perfusion measured in the skin area underinspection the blood supply of the tissue beneath the sensor membranemay, be inferred, the high resolution of the local and temporalcorrelation between perfusion and tcPO₂ or O₂-flux offering acomprehensive picture of the decisive parameters (perfusion and oxygenconcentration). Combination of these parameters will permit a newquality of medical diagnosis, which to date has been limited by theseparate use of the methods described as a combined process above, orother, previous measurement processes.

In a variant of the invention a photometric or photographic method maybe used as a second or further measuring process, where the measurementarea is recorded as an image in a predefined range of wavelengths. Inthis way autofluorescence or infrared radiation may be imaged in themeasured area. By means of infrared photography local heat distributionof a skin area may be detected as additional information.

Whereas in conventional luminescence-optical processes an opticallyisolating layer is usually applied between the skin and the sensormembrane for optical decoupling of emission radiation and backgroundfluorescence, the present invention features the use of a transparentsensor film, so that other measures must be taken to exclude stray lightcomponents during measurement. One possibility would be to detectluminophores with a long decay time (e.g., phosphorescent porphyrins ortransition metal complexes), once the short-lived luminescence ofinterfering substances (e.g., melanine, haemoglobin) in the sample hasbeen quenched. For signal separation it would also be possible to usevarious phase techniques, however.

It is provided in a further variant that a profilometric orinterferometric method be employed as a second or further measuringprocess. This will permit the topography of an organ or skin area to beincluded in the measurement in addition to the first measured value.Detecting the three-dimensional structure of the measured area is ofsome bearing for diagnosis and therapy, especially in the field ofdermatology or plastic surgery. Besides interferometric methods a stripeprojection method may be used, where a parallel ruled grating isprojected on the surface to be measured and evaluated by means of a CCDcamera placed at a certain angle relative to the projection plane. Smalldifferences in height of the inspected profile produce a distortion ofthe striped pattern which can be evaluated quantitatively.

The measured area could also be assessed visually through the sensorfilm (for example, by processes of epiluminescence microscopy). If atransparent sensor film is used the measured area may be assessed byvisual inspection, and morphological changes, such as colour changes ortumor growths may be directly correlated to luminescence data of thefirst measuring process. It would further be possible to put markings onthe skin surface which could be recorded visually or photographicallythrough the transparent sensor film.

In further development of the invention it is provided that errors inthe measured results of the second optical measuring process, which aredue to the sensor film, be corrected by a computing procedure orsuitable calibrating parameters. Radiation emitted by the LDI unit issubject to slight attenuation and scattering as it passes through thesensor film. If the entrance vector of the LDI beam is not normal to theboundary surface of the sensor film (due to the curved surface of themeasured object), a slight lateral displacement of the radiation pathwill result, which may be neglected on account of the low thickness ofthe sensor film of about 50 μm. On the other hand a certain part of theradiation will also be scattered during its passage from the skinthrough the sensor film to the LDI detector. As a consequence, themeasured value will further deviate from the value that would beobtained if no sensor film were present. By means of suitablecorrections using a computing procedure or stored calibration values itwill be possible to largely eliminate the influence of the sensor film.

LDP measurements often take several minutes for complete scanning of themeasurement area, depending on the desired resolution. This will alsolimit temporal resolution of the overall system. FLIM measurements needconsiderably less time (split seconds). To obtain a correlation of LDIand FLIM images of one and the same measurement area that issatisfactory from the aspect of time, every LDI measurement may bepreceded and followed by a FLIM measurement.

As an alternative, LDI and FLIM measurements could be “interlocked”,either by a short interruption of the LDI scan and fitting in asplit-second FLIM measurement, or by using the short time needed forrepositioning the laser beam onto a new measuring point, for performinga FLIM measurement. The lightsources for LDI and FLIM measurements maybe operated alternatingly, since the radiation detectors may be madesensitive to the emission radiation of both measurement processes.

In principle it will be possible to perform LDI and FLIM measurementssimultaneously. In this case separation of the detected signals must beensured. This is achieved either by spectral differentiation between LDIsignal and luminescence signal and corresponding selection of filters,or by selective electronic filtering of unmodulated and high-frequencymodulated signals.

The invention will be explained in more detail bellow, with reference tothe schematical drawings enclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a first variant of a system in accordance with theinvention,

FIG. 2 a detail of the system shown in FIG. 1,

FIG. 3 a very simple design variant, and

FIGS. 4 to 9 other advantageous variants of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIGS. 1 and 2 give a schematic view of a first variant of the systemproposed by the invention (a combination of FLIM and LDI), which can beused for simultaneously determining oxygen concentration and perfusionrate for a given measurement area m with high local resolution. Theexemplary measuring system essentially comprises a transducer (sensorfilm 7), a fluorescence lifetime imaging unit (FLIM), and aLaser-Doppler imaging unit (LDI).

-   1. Sensor film: The sensor film 7 shown in FIG. 2 has a transparent    support 14 as well as an indicator layer 13 on the side facing the    skin surface 8. The indicator layer 13 contains a luminescence    indicator, whose fluorescence quenching is uniquely defined by local    O₂ concentration. The indicator layer 13 should exhibit a sufficient    degree of absorption of the excitation radiation 11 emitted by the    FLIM unit, whereas the entire sensor film 7 including the support 14    is largely transparent to excitation and emission radiation 9, 10 of    the LDI unit 5, 6. Between the skin surface 8 and the indicator    layer 13 an adhesive layer 15 may be provided.    -   A typical variant of the sensor film features a flexible and        transparent polymeric multi-layer system, starting with a        support 14, for example of polyester, polyethylene, cycloolefin        copolymer, or a fluoropolymer, and a flexible indicator layer 13        that is essentially transparent to the emission radiation of the        additional measuring process. The indicator layer 13 contains a,        possibly oxygen-sensitive, luminscent dye, such as a        ruthenium-diimine-complex, an osmium-diimine complex, a platinum        porphyrin or palladium porphyrin, which is immobilized in a        polymeric matrix, e.g., silicone (with or without silica gel        fillers), polyvinyl chloride (PVC) with plasticizer,        polymethacrylate (PMMA), or polystyrene (PS).    -   According to another variant sensors for pH or CO₂ levels in the        skin may be used, which are based on other indicators, such as        hexapyrene-trisulphonic acid, or naphthalimide.    -   A third polymeric layer of the multi-layer system, which acts as        boundary between the sensor and the epidermis, preferably is        designed as adhesive layer 15, thus permitting continuous        contact with the skin surface and perfect gas diffusion between        skin and sensor. This adhesive layer may consist of a mixture of        silicone resin and uncured silicones (pressure-sensitive        adhesive, PSA).    -   In a variant as shown in FIG. 6 or 9 the matrix immobilizing the        dyes could be designed as adhesive layer itself, for example, by        incorporating a PSA into the matrix, or employing the PSA itself        as matrix for the dye.-   2. FLIM unit: This unit includes all mechanical, electronic and    optical components necessary for reproducible areawise excitation of    the indicator layer 13 and site-selective areawise detection of the    radiation 12 emitted by the indicator layer 13. As excitation    lightsource 1 a blue light-emitting diode may be used, which applies    the excitation radiation 11 via an excitation filter 2 on the sensor    film 7. The excitation radiation 11 must be able to pass the    essentially transparent support 14 while it should be, at least    partially, absorbed by the indicator layer 13 of the sensor film 7.    The luminescence radiation 12 coming from the indicator layer 13 is    detected via an emission filter 3 by a detection unit 4, preferably    a special CCD camera.    -   The spectroscopic data of an oxygen measurement are given as an        example, where the indicator (ruthenium-diimine complexes) is        excited by a lightsource (blue LED), the light of which is        partly absorbed by the indicator. The absorption maximum of the        indicator is situated at wavelengths of 460–490 nm. The emission        maximum of the indicator is between 580 and 630 nm. Maximum        optical density of the indicator layer in the wavelength range        of 430–480 nm is between 0.05 and 0.5.-   3. LDI-unit: This unit includes all mechanical, electronic and    optical components necessary for scanning the measurement area m    covered by the sensor film 7 with excitation radiation 9 through the    sensor film 7, and for detecting and analyzing emission radiation    10, and possibly correcting the measured values with regard to    scattering characteristics of the sensor film 7. A laser 5 supplies    monochromatic radiation of a wavelength that will practically not be    subject to absorption by the sensor film 7. Excitation radiation 9    will thus be maintained virtually unattenuated for analysis of the    biological sample, for example, the epidermis 8. Emission radiation    10, which is backscattered or reflected by the biologial sample 8,    will contain information that can be received and evaluated by the    detector 6 of the LDI unit. FLIM and LDI unit have a common input    and evaluation unit 16.

In all other variants components of the same kind or function have thesame reference numerals.

In FIG. 3 a very compact variant of the invention is shown, in which thesecond optical measuring device is provided with a unit 17 forseparation of radiations 10, 12 emitted by the two optical measuringdevices, such as a filter wheel with different emission filters 3, 3′for the two radiations 10, 12. The system has only one lightsource 1 andone CCD camera. This variant is particularly well suited if the FLIMprocess is combined with reflexion spectrophotometry orauto-fluorescence measurement.

FIGS. 4 to 6 show systems where the FLIM process is combined withreflexion-spectrophotometry. In FIG. 4 a broad-band lightsource is usedas separate excitation lightsource 5, whose excitation radiation 9 isdirected onto the sample 8 via an excitation filter 2′. For the purposeof measurement the CCD camera 4 of the FLIM process is used, which isfed with the two emission radiations 10, 12 via emission filters 3, 3′of the filter wheel 17. The variant of FIG. 5 differs from that of FIG.4 by the use of a separate imaging detection unit 6 (such as a secondCCD camera), which is supplied with the emission radiation 10 ofreflexion spectrophotometry. FIG. 6 shows the interaction betweenexcitation radiation 11 (FLIM) and indicator layer 13, and thescattering of excitation radiation 9 in the epidermis 8.

FIGS. 7 to 9 show systems where the FLIM process is combined withautofluorometry. According to FIG. 7 a laser is used as separateexcitation lightsource 5, whose light is directed onto the sample 8 bymeans of a beam expander. Emission radiation 10 may be detected via afilter wheel 17 by the CCD camera 4 (FIG. 7), or by a separate, imagingdetection unit 6 (FIG. 8). FIG. 9 shows the interaction betweenexcitation radiation 11 (FLIM) and indicator layer 13, and that ofexcitation radiation 9 (autofluorescence) in the epidermis 8.

1. Method for determining by imaging techniques the local distributionof first and second independent measurement variables measured relativeto or present in a predefined measurement area of a biological samplecomprising the steps of: applying a single sensor film to the predefinedmeasurement area of the biological sample, said single sensor filmcontaining a luminescence indicator which reacts to said firstindependent measurement variable by a change in luminescence decay time,directing first excitation radiation into said single sensor film toexcite said luminescence indicator and cause said luminescence indicatorto emit first measurement radiation, detecting said first measurementradiation and determining therefrom local distribution of said firstindependent measurement variable in a first optical measuring process,directing second excitation radiation into and through said singlesensor film and into said biological sample to cause second measurementradiation to be emitted from said biological sample, and detecting saidsecond measurement radiation and determining therefrom localdistribution of said second independent measurement variable in a secondoptical measuring process.
 2. Method according to claim 1, wherein saidbiological sample is the epidermis or the surface of an organ.
 3. Methodaccording to claim 1, wherein said first measurement variable to bedetermined is one of the group consisting of the local distribution ofoxygen concentration (tcPO₂), the local distribution of transcutaneousoxygen concentration and the local distribution of oxygen flow (O₂-flux)through an organ surface.
 4. Method according to claim 3, wherein saidorgan surface is skin.
 5. Method according to claim 1, wherein saidfirst measurement variable determined is the local distribution of CO₂concentration or the local distribution of CO₂ flow.
 6. Method accordingto claim 1, wherein said first measurement variable determined is thelocal distribution of one of the group consisting of temperature,glucose concentration and an ionic concentration.
 7. Method according toclaim 6, wherein said ionic concentration is the pH level.
 8. Methodaccording to claim 1, wherein said second measuring process is a LaserDoppler imaging (LDI) process being used to determine a perfusion ratein said measurement area as said second measurement variable.
 9. Methodaccording to claim 1, wherein said second measuring process is aphotometric or photographic method and said measurement area is recordedas an image in a predefined range of wavelengths.
 10. Method accordingto claim 9, wherein autofluorescence in said measurement area isrecorded as said image.
 11. Method according to claim 9, whereininfrared radiation in said measurement area is recorded as said image.12. Method according to claim 1, wherein said second measuring processis a profilometric method.
 13. Method according to claim 12, whereinsaid profilometric method is an interferometric method.
 14. Methodaccording to claim 1, wherein said second measuring process is a visualinspection of said measurement area being assessed through said sensorfilm.
 15. Method according to claim 1, wherein errors in measuredresults of said second optical measuring process, which are due to saidsensor film, are corrected by a computing procedure or suitablecalibrating parameters.